An Improved Agrobacterium-Mediated Transformation Protocol for Recalcitrant Elite Indica Rice Cultivars Essay

Plant Molecular Biology Reporter twenty-three: 67–73, Drive 2005 © 2005 Intercontinental Society for Plant Molecular Biology. Imprinted in Canada.

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An Improved Agrobacterium-Mediated Transformation Process for Recalcitrant Elite Indica Rice Cultivars K. E. KUMAR, S. MARUTHASALAM, M. LOGANATHAN, M. SUDHAKAR and P. BALASUBRAMANIAN* Rice Transformation Laboratory, Department of Herb Molecular Biology and Biotechnology, Center for Plant Molecular Biology, Tamil Nadu Agricultural University, Coimbatore-641003, Tamil Nadu, India Fuzy. We survey here a high-efficiency transformation protocol pertaining to recalcitrant indica rice cultivars IR64 and IR72 together with the selectable marker gene hph and the apetito reporter gene. Factors that favor high-efficiency transformation had been found to get use of 2-month-old mature seed-derived embryogenic calli, maltose like a source of carbon, a higher concentration of 2, 4-dichlorophenoxyacetic acid, and both phytagel and agar agar as solidifying agents. The putative transgenic (T0) vegetation were examined for integration of the transgene through polymerase chain effect and The southern area of blotting examines. Various factors thought to be accountable for increased change efficiency happen to be discussed. Keywords and phrases: Agrobacterium, arata rice, adult seed-derived calli, rice change, transgenic grain Abbreviations: a couple of, 4-D, two, 4-dichlorophenoxyacetic acidity; BAP, 6-benzylaminopurine; MS, Murashige and Skoog; NAA, О±-naphthaleneacetic acid; OD, optical thickness; PCR, polymerase chain effect. Agrobacterium-mediated modification protocol intended for indica Advantages rice cultivars Kumar ain al.

Agrobacterium-mediated transformation is routinely intended for the transformation of many monocots, including grain. Several reviews are available around the Agrobacterium-mediated alteration of japonica and indica rice cultivars. However , the efficiency with which the indica rice cultivars are transformed is very low. Different explant types are used in the Agrobacterium-mediated transformation of rice, which include mature seed-derived calli, premature embryo-derived calli, immature embryos, and shoot apex (Hiei et ing., 1994; Aldemita and Hodges, 1996; Playground et al., 1996). Though Aldemita and Hodges (1996) obtained comparatively high modification efficiency for indica rice by using premature embryos pertaining to cocultivation, this method has many restrictions, such as non-availability throughout the year and laboriousness. Hiei et approach. (1994) and Rashid et al. (1996) *

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Kumar ainsi que al.

reported successful transformation of japonica and easily responsive indica grain genotypes by using mature seed-derived calli while explants and found that 3-week-old mature seed-derived calli will be the most suitable explants for Agrobacterium-mediated transformation. There are several reports about transformation of recalcitrant arata rice cultivars, for example , IR64 and IR72, through Agrobacterium (Datta ou al., 1999, 2000, 2001; Khanna and Raina, 1999, 2002; Tu et approach., 1998; Zhang et al., 1998), having a relatively low frequency of transformation. We report here an improved transformation protocol to get 2 top notch indica rice cultivars, IR64 and IR72, by use of mature seed-derived calli since explants. Components and Strategies Plant supplies used Fully developed seeds of rice cultivars IR64 and IR72 (obtained from Terme conseille Breeding Train station, Tamil Nadu Agricultural University, Coimbatore) were manually dehusked, after which they were sterilized with 70% ethanol for a few min after which with zero. 1% mercuric chloride for 5 min, followed by 3 washes in sterile distilled water. Sterilized seeds had been cultured in callus debut ? initiation ? inauguration ? introduction medium (MS salts [Murashige and Skoog, 1962] that contain B5 nutritional vitamins, 30 g/L maltose, 3. 0 mg/L 2, 4-dichlorophenoxyacetic acid [2, 4-D], 0. a few mg/L kinetin, 4. 0 g/L phytagel, and 2 . 0 g/L) agar for 25В°C in the dark. After 3-4 wk, the proliferating calli were subcultured onto similar...

References: Aldemita PR and Hodges TK (1996) Agrobacterium tumefaciens–mediated alteration of japonica and indica rice types. Planta 199: 612-617. Chilton MD, Currier TC, Farrand SK, Bendich AJ, Gordon MP, and Nester EW (1974) Agrobacterium tumefaciens GENETICS and P58 bacteriophage DNA not recognized in overhead gall tumours. Proc Natl Acad Sci USA 71: 3672-3676. Datta K, Koukolikova-Nicola Z, Baisakh N, Oliva N, and Datta SK (2000) Agrobacterium-mediated engineering to get sheath blight resistance of indica rice cultivars via different ecosystems. Theor Appl Genet 90: 832-839. Datta K, Tu J, Oliva N, Ona I, Velazhahan R, Mew TW, Muthukrishnan S, and Datta SK (2001) Enhanced resistance to sheath blight simply by constitutive phrase of infection-related rice chitinase in transgenic elite arata rice cultivars. Plant Sci 60: 405-414. Datta, E, Velazhahan 3rd there’s r, Oliva N, Ona I actually, Mew Capital t, Kush GS, Muthukrishnan S, and Datta SK (1999) Over-expression with the cloned rice thaumatin-like necessary protein (PR-5) gene in transgenic rice plants enhances environmental friendly capacity Rhizoctonia solani causing sheath blight disease. Theor Appl Genet 98: 1138-1145. Dellaporta SL, Solid wood J, and Hicks JB (1983) A plant GENETICS minipreparation: variation II. Grow Mol Biol Rep you: 19-21. Ditta G, Stanfield S, Corbin D, and Helinski M (1980) Broad host selection DNA cloning system pertaining to gram-negative bacterias: construction of the gene lender of Rhizobium meliloti. Proc Natl Acad Sci UNITED STATES 77: 7347-7351. Hiei Y, Ohta S, Komari Capital t, and Kumashiro T (1994) Efficient transformation of rice (Oryza

Agrobacterium-mediated transformation protocol for arata rice cultivars

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sativa L. ) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant L 6: 271-282. Jain RK, Davey MR, Cocking EC, and Wu R (1997) Carbohydrate and osmotic requirements for high-frequency plant regeneration from protoplast-derived colonies of indica and japonica rice varieties. J Exp Botany 48: 751-758. Khanna HASTKRAFTER and Raina SK (1999) Agrobacterium-mediated change of arata rice cultivars using binary and superbinary vectors. Aust J Flower Physiol 26: 311-324. Khanna HK and Raina SK (2002) High level indica transgenic rice plant life expressing revised Cry1A(c) endotoxin of Bacillus thuringiensis confirmed enhanced capacity yellow control borer (Scirpophaga incertulas). Transgenic Res 10: 411-423. Murashige T and Skoog FA (1962) A revised medium for speedy growth and bioassays with tobacco muscle cultures. Physiol Plant 15: 473-497. Playground SH, Pinson SRM, and Smith RH (1996) T-DNA integration in to genomic GENETICS of rice following Agrobacterium inoculation of isolated blast apices. Herb Mol Biol 32: 1135-1148. Rashid H, Yokoi S i9000, Toriyama K, and Hinata K (1996) Transgenic plant production mediated by Agrobacterium in indica rice. Grow Cell Repetition 15: 727-730. Roberts-Oehlschlager SL, Dunwell JM, and Faulks R (1990) Changes in glucose content of barley anther during lifestyle on several carbohydrates. Grow Cell Tissues Organ Conspiracy 22: 77-85. Sambrook J, Fritsch EF, and Maniatis T (1989) Molecular Cloning: A Lab Manual, second ed. Cool Spring Harbor Laboratory Press, Cold Planting season Harbor, New york city. Toriyama E and Hinata K (1985) Cell postponement, interruption and protoplast culture in rice. Flower Sci forty one: 179-183. Su J, Ona I, Zhang Q, Mew TW, Khush GS, and Datta SK (1998) Transgenic rice variety IR72 with Xa21 can be resistant to bacterial blight. Theor Appl Genet 97: 31-36. Zhang S, Song WY, Chen D, Ruan M, Taylor D, Ronald LAPTOP OR COMPUTER, Beachy RN, and Fauquet CM (1998) Transgenic high level indica rice varieties, immune to Xanthomonas oryzae pv. oryzae. Mol Breed of dog 4: 551-558.



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